Gene expression is mediated by DNA- and RNA-binding proteins in every organism. Regulation of gene expression is commonly mediated by protein modifications of these nucleic acid binding proteins. This proposal focuses on an under-explored but therapeutically important protein modification called poly(ADP- ribose) (PAR). PAR has been well known for its roles in DNA repair and transcription in the nucleus. Recently, we discovered that PAR also modifies several post-transcriptional mRNA gene regulators in the cytoplasm. Our data are consistent with recent proteomics studies showing that poly(ADP-ribosyl)ated (PARylated) proteomes are enriched with RNA-binding proteins, suggesting that PAR plays a much broader regulatory role in RNA metabolism than previously appreciated. In this proposal, we will focus on how PAR regulates microRNA functions. MicroRNAs are a class of ~22 nucleotide non-coding RNAs that regulate many fundamental cellular processes, including stress responses. Although much has been characterized about microRNA biogenesis, little is known about how microRNA activities are regulated. Key data: Recent data including ours indicate that microRNA activities are inhibited by the PARylation of the core microRNA-binding protein Argonaute (AGO). Such inhibition is regulated by PAR polymerase 13 (PARP- 13) where its overexpression reduces microRNA activities. Intriguingly, PARP-13 is catalytically inactive; therefore, other catalytically active PARP(s) must be involved. Such a PARylation mechanism involving more than one PARP represents a new paradigm. In this proposal, we will investigate how PARP-13 interacts with a cytoplasmic, catalytically active PARP to PARylate AGO (Aim 1), determine how PAR polymers on AGO reduce microRNA activities (Aim 2) and identify which domains of AGO are modified by PAR (Aim 3). We will use a novel mass spectrometry technique to identify AGO PARylation sites. Until now, the identification of PARylation sites has been a challenge for the field and thus this study allows the first systematic analysis of the functional roles of individual sites of a protein substrate. O note, PARP-13 is also known as zinc antiviral protein (ZAP) - a host factor that inhibits the replication of Sindbis virus, Ebola virus, Hepatitis B virus and HIV upon overexpression. Therefore, AGO PARylation-mediated inhibition of microRNA activities may be involved in host antiviral responses, which we will explore in the context of Sindbis virus infection (Aim 3c) in collaboration with Dr. Diane Griffin at Johns Hopkins. The Team: To ensure success, this project is performed with two key collaborators (both of whom we are requesting for one funding module): AGO biochemistry expert Dr. Leemor Joshua-Tor (Cold Spring Harbor Laboratory) and proteomics expert Dr. Shao-En Ong (University of Washington). Other consultants include Drs. Phillip Sharp (MIT) and Carl Novina (Harvard) on microRNA biology, Drs. Ted Dawson (John Hopkins) and Paul Chang (MIT) on PAR biology, and Dr. Pierre Coulombe (Johns Hopkins) on biochemistry.